ELECTRON TOMOGRAPHY OF VERTEBRATE MITOTIC SPINDLES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 将使用快速冷冻、冷冻置换固定样品的电子断层扫描检查U2 OS细胞中期纺锤体体积的显著部分，然后对纺锤体形成期间冷冻和固定的细胞进行类似工作。 我们的结构研究将采用改进的样品制备方法（TR&D 3A）和大规模蒙太奇和多个连续切片，组装成3D重建（TR&D 5D）。纺锤体微管（MT）的轨迹最初将通过手工建模来确定，但在随后的工作中，我们希望有自动分割的软件。将使用聚类分析和几种特定于应用的软件工具来研究MT轨迹的结果模型，以将MT组织与四种不同且流行的纺锤体形成模型的预测进行比较：经典的中心体起始模型，认为纺锤体MT的自我组织是MT相关的运动酶的结果，MT起始和/或稳定的想法，响应于从染色体发出的梯度，以及非MT“基质”指导MT的形成和组织的概念。 这些数据应该消除或至少削弱这些模型中的一个或多个，并且肯定会为我们实验室和其他地方正在进行的关于纺锤体形成和功能的功能实验的解释提供基线结构信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Electron tomography of rapidly frozen, freeze-substitution fixed samples will be used to examine a significant fraction of the volume of metaphase spindles from U2OS cells, followed by comparable work on cells frozen and fixed during spindle formation. Our structural studies will employ improved methods for sample preparation (TR&D 3A) and both large-scale montages and multiple serial sections, assembled into 3D reconstructions (TR&D 5D). The trajectories of spindle microtubules (MTs) will initially be determined by hand modeling, though in subsequent work we hope to have software for automatic segmentation. The resulting models of MT trajectories will be studied with cluster analysis and several application-specific software tools to compare MT organization with the predictions from four distinct and popular models for spindle formation: the classic, centrosome-initiation model, the idea that spindle MTs self-organize as a result of MT-associated motor enzymes, the idea of MT initiation and/or stabilization in response to gradients that emanate from chromosomes, and the concept that a non/MT "matrix" guides the formation and organization of MTs. The data should eliminate or at least erode one or more of these models and will certainly provide baseline structural information for the interpretation of functional experiments on spindle formation and function that are being carried out in our labs and elsewhere.